The resulting recombinant plasmids were sequenced using a pMD20-T F13 primer (5-TTCGAGCTCGGTACCCGGGGATCCGATT-3) and pMD20-T F47 primer (5-AATCCATATGACTAGTAGATCCTCTAG-3). culture samples were collected at every time point, and the viral replication dynamics (Fig.?1b) were determined by one-step real-time quantitative RT-PCR (one-step qRT-PCR). The viral RNA load was still very low at 12?h.p.i. , about 102 to 105, and it had reached 107 at 24 and 48?h.p.i. The BHK21-P cells had a significantly lower viral RNA load than the BHK21-N cells at all of the selected time points after infection with 100 TCID50 FMDV (transgenic cloned swine group, non-transgenic cloned swine group, normal control swine Calcipotriol monohydrate group, vaccinated swine group. (a) The development of clinical symptoms and clinical lesion score. Green: healthy; yellow: ill (score below 24); orange: seriously ill (score above 24); black: dead; blue: recuperated and healthy. The number in the column for days post-challenge represents the clinical score, – indicates that the clinical score was the same as the last time point. (b) Mean onset of lesions (day) in the unprotected swine. (c) Mean time of developing severe lesions (days) in the unprotected swine. (d) Mean onset of viremia in the unprotected swine (d). (e) Mean duration of viremia in the unprotected swine. #Swine developed lesions/Swine challenged with virus. ^Swine developed viremia/Swine challenged with virus. *indicates a polyA polymerase, and then the PolyA reaction solution was used for the first-strand cDNA synthesis using the miRcute miRNA First-strand cDNA Synthesis kit (TIANGEN) protocol. Real-time PCR was performed using an miRcute miRNA qPCR Detection kit (TIANGEN) on an MX3000p system from Stratagene, and the data were analyzed using the MX3000p software provided by the manufacturer. Each reaction was run in triplicate. We used U6 as the internal DNA control, the primers was designed for 2B siRNA and U6. The primers used for the experiment were as follows: 2BshRNA_F: 5-GTCACCAGATGCAGGAGGATATG-3; U6_F: 5-CGCAAATTCGTGAAGCGTTC-3. All of the reverse primers were provided with the kit. Generation and identification of the shRNA transgenic cloned swine The linearized p3DEN2B construct (the same with the constuct used in cell lines) was transfected into the swine fibroblast cell line using the Amaxa Nucleofector? II Device. The transfected fibroblasts were cultured and passaged in selected culture medium with G418 (400?g/mL, Promega) for fourteen days. The antibiotic-resistant colonies were selected and identified by PCR to confirm that the p3DEN2B vector had been inserted. The fibroblasts containing the vector were used as the nuclear donors in the somatic nuclear transfer (SCNT) procedure. SCNT was performed as previously described66 (Fig.?2). Viral challenge of the swine and clinical analysis Ten transgenic cloned swine and five non-transgenic cloned swine were transported Calcipotriol monohydrate to the National P3 Lab at the Lanzhou Veterinarian Research Institute for the challenge experiments. Calcipotriol monohydrate The cloned swine were all 3-month-old Landrace male swine weighing ca. 40C50?kg. The wild-type swine were used to perform a viral challenge as the normal control and vaccine-inoculated control. None of the swine had previous FMDV contact, as confirmed by the absence of detectable anti-FMDV antibodies (use LPBE as below) or antibodies against other viruses, such as porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and porcine circovirus 2 (PCV2), in their serum. The virus CHA/86 used to challenge was titrated in wild-type swine. The dose of FMDV used was determined by means of four tenfold serial dilutions of the virus (i.e., 10-4, 10-5, 10-6, and 10-7) in phosphate-buffered saline (PBS). Calcipotriol monohydrate Sixteen swine were divided into four groups (four swine/each group). Each animal was inoculated in the neck region by intramuscular injection of 3?mL of the serially diluted virus. All of the animals were monitored twice per day for the major clinical symptoms of FMD, namely, mouth and foot lesions. The 50% swine infective dose (SID50) was estimated as 6.0/3?mL according to the Reed-Muench method (Reed et al. 1938). All of the animals were housed in disease-security isolation facilities in TMOD4 the P3 Lab. Twelve control swine were inoculated with 2?mL of vaccine (Swine Foot and Mouth Disease Type O vaccine, Inactivated II, China Agricultural Vet. Bio. Science and Technology Co., Ltd.) by intramuscular injection in the neck area three weeks before the viral challenge. Ten swine were randomly chosen as the vaccinated controls. The swine without vaccination was used as the normal swine controls. Ten transgenic cloned swine, 10 wild-type swine, and 10 vaccine immunized swine were equally divided into two challenge studies (100 SID50 and 10 SID50). All animals were challenged by intramuscular injection with 3?mL of 100 SID50 or 100 SID50 of CHA/86. After the challenge, the swine that developed the disease were.